1. Field of the Invention
This invention relates to the application of small volumes of reagents to surfaces. In one aspect the invention relates to the manufacture of arrays formed and arranged by depositing compounds or synthesizing large numbers of compounds on solid supports in a predetermined pattern. In another aspect this invention relates to the field of bioscience in which arrays of oligonucleotide probes are fabricated or deposited on a surface and are used to identify or analyze DNA sequences in cell matter. The present invention has a wide range of application for synthesis and use of arrays of oligonucleotides or proteins for conducting cell study, for diagnosing disease, identifying gene expression, monitoring drug response, determination of viral load, identifying genetic polymorphisms, and the like.
Significant morbidity and mortality are associated with infectious diseases and genetically inherited disorders. More rapid and accurate diagnostic methods are required for better monitoring and treatment of these conditions. Molecular methods using DNA probes, nucleic acid hybridization and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double-stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the development of methods for their incorporation into DNA and RNA has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses.
The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited due to the cost and effort associated with the development of sufficiently sensitive and specific methods for detecting potentially low concentrations of disease-related DNA or RNA present in the complex mixture of nucleic acid sequences found in patient samples.
One method for detecting nucleic acids is to employ nucleic acid probes that have sequences complementary to sequences in the target nucleic acid. A nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support. Detection of signal depends upon the nature of the label or reporter group. Usually, the probe is comprised of natural nucleotides such as ribonucleotides and deoxyribonucleotides and their derivatives although unnatural nucleotide mimetics such as 2xe2x80x2-modified nucleosides, peptide nucleic acids and oligomeric nucleoside phosphonates are also used. Commonly, binding of the probes to the target is detected by means of a label incorporated into the probe. Alternatively, the probe may be unlabeled and the target nucleic acid labeled. Binding can be detected by separating the bound probe or target from the free probe or target and detecting the label. In one approach, a sandwich is formed comprised of one probe, which may be labeled, the target and a probe that is or can become bound to a surface. Alternatively, binding can be detected by a change in the signal-producing properties of the label upon binding, such as a change in the emission efficiency of a fluorescent or chemiluminescent label. This permits detection to be carried out without a separation step. Finally, binding can be detected by labeling the target, allowing the target to hybridize to a surface-bound probe, washing away the unbound target and detecting the labeled target that remains.
Direct detection of labeled target hybridized to surface-bound probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas of the surface. Such ordered arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations.
In one approach, cell matter is lysed, to release its DNA as fragments, which are then separated out by electrophoresis or other means, and then tagged with a fluorescent or other label. The resulting DNA mix is exposed to an array of oligonucleotide probes, whereupon selective attachment to matching probe sites takes place. The array is then washed and imaged so as to reveal for analysis and interpretation the sites where attachment occurred.
In the preparation of arrays, reagents are applied to predetermined discrete locations on the surface of a substrate. Depending on the type of synthesis and array, the preparation may involve application of reagents at discrete locations followed by treatment of a portion or the entire surface with a different liquid reagent. The steps may be repeated a number of times sufficient to prepare the desired array. Examples of known methods for subjecting all or a portion of substrate surfaces to reagents include flooding, spin coating and flow cell assembly. Flooding the surface may be accomplished by using, for example, a multi-nozzle piezoelectric pump head. A relatively large volume of liquid is dispensed to contact the surface and assure that the dispensed reagents contact all of the desired locations. Spin coating is usually performed by dispensing the reagent at or near the center of the substrate followed by spinning to spread the reagent uniformly across the substrate.
The volume used to cover the substrate depends on the fluid property and the surface energy of the substrate. Some approaches used for in situ synthesis require a large relative volume to cover the surface because small, dispensed volumes tend to cluster and non-uniformly cover the surface. The reagent is then removed from the substrate within a high-speed spin step, which generates a considerable amount of waste. In the flow cell approach, a seal layer is brought in contact with the substrate at various support points (typically along the perimeter). A thin gap exists between the substrate and seal layer. By developing a pressure gradient across inlet and outlet channels, fluid can be forced to flow in the gap along the substrate. Although this method can use considerably less volume than the flooding method or the spin coat method, it has three major drawbacks. First, there is a long fill time in order to support laminar flow. Second, it is prone to leaking if uniform pressure is not maintained. Third, variability in surface thickness will disturb the laminar flow resulting in air pockets and hence non-uniform coverage.
2. Description of the Related Art
U.S. Pat. No. 5,831,070 (Pease, et al.) discloses printing oligonucleotide arrays using deprotection agents solely in the vapor phase.
Sono-Tek Corporation brochure, copyright 1996.
One embodiment of the present invention is a method for conducting chemical reactions on a surface comprising an array of discrete sites. Molecules are deposited at a predetermined number of the discrete sites on the surface for reaction at the discrete sites. The surface is positioned relative to an outlet of a fluid ejection device, which is activated to dispense a small volume of a fluid through the outlet to the surface to provide uniform coating of a continuous region of the surface comprising a multiple of the discrete sites. The fluid is dispensed as uniform particles having a diameter such that the uniform particles form a sheet to coat the continuous region of the surface.
Another embodiment of the present invention is a method for forming an array of molecules at discrete sites on a surface. Molecule precursors are applied to predetermined discrete sites on the surface. A small volume of a liquid is dispensed to uniformly coat a continuous region of the surface comprising a multiple of the discrete sites. The small volume of liquid is dispensed as uniform particles through a fluid ejection device activated by means of ultrasonic energy.
Another embodiment of the present invention is a method for forming an array of polynucleotides at discrete sites on a surface. Reagents are applied to predetermined discrete sites on the surface. The reagents are selected from the group consisting of nucleotides and polynucleotides. A volume of a liquid is dispensed to uniformly coat the surface with liquid. The volume of liquid is dispensed as particles of uniform diameter through a fluid ejection device activated by means of ultrasonic energy. Step (a) or step (b) may optionally be repeated.
Another embodiment of the present invention is a method for forming an array of polynucleotides at discrete sites on a surface. Nucleotide reagents are applied to predetermined discrete sites on the surface. A volume of a liquid of about 1 nanoliter to about 1000 nanoliters is dispensed to uniformly coat the surface with liquid. The liquid is dispensed as particles of uniform diameter of about 1 microns to about 500 microns through a fluid ejection device activated by means of ultrasonic energy at a frequency of about 5 kilohertz to about 300 kilohertz. The liquids comprise agents selected from the group consisting of wash liquids, deblocking agents and deprotection agents. Step (a) or step (b) optionally may be repeated.
Another embodiment of the present invention is an apparatus for forming an array of polynucleotides at discrete sites on a surface. The apparatus comprises a device for dispensing reagents to predetermined discrete sites on said surface and a fluid ejection device activated by means of ultrasonic energy. The fluid ejection device dispenses a volume of a liquid as particles of uniform diameter to uniformly coat the surface with liquid. The reagents are selected from the group consisting of nucleotides and polynucleotides.